We describe here the characterization of eight different fully human IgGs directed to SARS-CoV that were isolated from semisynthetic human antibody libraries. Since complete SARS-CoV virions, rather than a single recombinant protein or fragment thereof, were used as antigen for selections, MAbs against different proteins in their natural conformation were isolated. Target identification revealed that two MAbs reacted with the N protein, and EM performed with both MAbs enabled us to visualize the presence of N protein within virions produced by SARS-CoV-infected Vero cells.
The epitopes of these noncompeting MAbs, CR3009 and CR3018, were investigated in more detail by using Pepscan analysis. Through this approach, the minimal binding site of CR3018 was mapped to residues 11 to 19 of the N protein, which corresponds to the sequence RSAPRITFG. Interestingly, this linear epitope is conserved in the N protein sequence of all published human SARS-CoV and animal SARS-CoV-like isolates but is absent in other members of the family of Coronaviridae
. Assessment of antigenic peptides derived from SARS-CoV structural proteins revealed that 9 of 31 sera from SARS patients tested reacted with a peptide composed of residues 1 to 23 of N protein (41
). This indicates that a significant proportion of the SARS patients develops antibodies to N protein, which are directed to an epitope similar to that recognized by CR3018. Future studies should reveal the level of sequence homology between human MAbs isolated from the antibody repertoire of patients with SARS and antibody CR3018, which was isolated from a semisynthetic scFv phage display library. Epitope mapping of MAb CR3009 was unsuccessful, presumably because CR3009 recognizes a nonlinear epitope. Besides a large number of linear epitopes (16
), the N protein contains two major conformational epitopes recognized by the sera of SARS patients (5
). These characteristics of both CR3009 and CR3018 could be exploited in a diagnostic test specific for SARS-CoV.
At present, solid proof of SARS-CoV infection is provided after isolation of the virus from a clinical specimen, a confirmed positive PCR for SARS-CoV or detection of antibody seroconversion. Virus isolation is time-consuming, and PCR requires technical equipment, which is not available in every local hospital. In the majority of the patients, seroconversion is only detectable from the second or third week after disease onset (24
), making this late and retrospective diagnostic tool ineffective for quarantine measures. Furthermore, antibodies to SARS-CoV or related viruses have already been detected in blood samples taken from healthy individuals 2 years before the most recent SARS outbreak (30
). Taken together, these findings emphasize the need for an instant and more accurate laboratory test for the early diagnosis of SARS. MAbs that specifically detect SARS-CoV proteins may therefore greatly facilitate the development of a SARS-CoV-specific immunoassay.
In addition to N protein MAbs, four MAbs to the S protein were isolated, three of which were capable of effectively neutralizing SARS-CoV infectivity in vitro. The epitope of the nonneutralizing antibody, CR3015, is located outside the region comprising residues 1 to 565 and could be located within the S2 domain. Human antibodies binding to different epitopes in the S2 domain protein have been described previously (41
). Sui et al. reported previously eight scFv, all directed to the S1 domain (residues 1 to 672), of which only one, 80R, was capable of neutralizing SARS-CoV infectivity (38
). This indicates that not all antibodies binding to the S1 domain of the S protein do interfere with the infectivity of SARS-CoV. MAbs CR3006, CR3013, and CR3014 described here compete for binding to the S1 domain with different affinities and neutralize SARS-CoV. However, antibody affinity and neutralizing potency do not necessarily correlate. Traggiai et al. isolated two types of neutralizing MAbs with Epstein-Barr virus transformation. Some MAbs showed neutralizing titers proportional to their degree of binding, whereas others showed low-avidity binding in spite of efficient viral neutralization (40
We demonstrated that the epitopes of our neutralizing MAbs are located within the previously identified minimal ACE2 receptor-binding region of the S protein; a more precise characterization of the epitope by using Pepscan analysis failed. Most likely, MAb CR3014 recognizes a more complex conformational epitope within the S1 domain that cannot be detected by this technique. This suggests that the MAb CR3014 binding site is different from that of MAb 80R (38
), which was shown to remain partially intact under denaturing and reducing conditions. Also, deglycosylation of the S1 domain did not prevent R80 from binding to its epitope. Binding studies with variant recombinant S318-510 fragments revealed that the epitope of CR3014 is conserved in the S proteins of all human SARS-CoV isolates described in Table . Reduced reactivity with a variant S318-510 fragment harboring a N479S substitution suggests a substantial contribution of this residue to binding of CR3014. Residue N479 may either be directly involved in binding of CR3014 by being part of the antibody binding site or, alternatively, contribute to a correct conformation of the antibody binding site. The epitope of CR3006 was completely destroyed by the introduction of naturally occurring amino acid substitutions of residues Y442 or F360, L472, D480, and T487, as are present in two different SARS-CoV isolates. One of these isolates was collected in December 2003 from the last infected patient not related to a laboratory-acquired SARS infection (6
). These data illustrate the importance of evaluating the specificity of anti-S MAbs for a wide variety of SARS-CoV isolates.
The development of neutralizing antibodies in patients with SARS is similar to that observed in other acute viral infectious diseases such as hepatitis A (19
). The preventive value of IgG against hepatitis A infection was demonstrated as early as 1945 (36
), and prevention of rabies after exposure requires the administration of immunoglobulin prepared from hyperimmune sera in combination with vaccine (33
). Based on these observations and the successful use of a recombinant MAb against respiratory syncytial virus that prevents disease in high-risk infants (20
), immunoprophylaxis of SARS-CoV infection with MAbs might be an option for the control of SARS (18
To this end, we evaluated whether the neutralizing capacity of CR3014 in vitro can abolish the infectivity of SARS-CoV in ferrets, essentially as described by Emini et al., for infectivity of human immunodeficiency virus type 1 (HIV-1) in chimpanzees (11
). In this ferret model, infection of the animals via the intratracheal route leads to massive replication of the virus in the lung and the development of pulmonary SARS-CoV-associated lesions accompanied by various degrees of nonlethal clinical disease (28
). High virus titers were observed in the lungs of control ferrets on day 4, which dropped at day 7, thereby following the natural course of infection with SARS-CoV. Animals that received a combination of CR3014 and virus had almost undetectable titers of SARS-CoV in their lungs. In a follow-up study, we demonstrated that prophylactic administration of CR3014 at 10 mg/ml reduced replication of SARS-CoV in the lungs of infected ferrets, prevented SARS-CoV-induced macroscopic lung pathology, and abolished the shedding of virus in pharyngeal secretions (39
Thus, SARS-CoV neutralizing antibodies may be used to prevent infection in people exposed to the SARS-CoV, such as hospital personnel caring for suspected SARS patients, and may also be applied for the early treatment of infected individuals to avoid the onset of severe SARS disease and to lower the chance of spreading the virus to exposed individuals.